Vaccine co-display of CSP and Pfs230 on liposomes targeting two Plasmodium falciparum differentiation stages

A vaccine targeting multiple stages of the Plasmodium falciparum parasite life cycle is desirable. The sporozoite surface Circumsporozoite Protein (CSP) is the target of leading anti-infective P. falciparum pre-erythrocytic vaccines. Pfs230, a sexual-stage P. falciparum surface protein, is currently in trials as the basis for a transmission-blocking vaccine, which inhibits parasite development in the mosquito vector. Here, recombinant full-length CSP and a Pfs230 fragment (Pfs230D1+) are co-displayed on immunogenic liposomes to induce immunity against both infection and transmission. Liposomes contain cobalt-porphyrin phospholipid (CoPoP), monophosphoryl lipid A and QS-21, and rapidly bind His-tagged CSP and Pfs230D1+ upon admixture to form bivalent particles that maintain reactivity with conformational monoclonal antibodies. Use of multicolor fluorophore-labeled antigens reveals liposome binding upon admixture, stability in serum and enhanced uptake in murine macrophages in vitro. Bivalent liposomes induce humoral and cellular responses against both CSP and Pfs230D1+. Vaccine-induced antibodies reduce parasite numbers in mosquito midguts in a standard membrane feeding assay. Mice immunized with liposome-displayed antigens or that passively receive antibodies from immunized rabbits have reduced parasite liver burden following challenge with transgenic sporozoites expressing P. falciparum CSP.


Significance:
The article by Huang et al. provides detailed analysis of CoPoP liposomes admixed with two malaria antigens Pfs230 and PfCSP for their transmission/infection blocking activity. These liposomes are stable at 37C, confirmational integrity of antigens is intact and can elicit antigen specific cellular immune response in the animals. These finding represent significant advance by targeting more than one Plasmodium life cycle stages.

Comments:
Change "supporting" table# to "Supplementary "table throughout the manuscript. Supplementary Table 3. The mean/median oocyst numbers and % oocyst prevalence per for each feed in SMFAs should be provided for clarity. Figure 4 and Supplementary Table 3. Only 20 mosquitoes were dissected per experiment in SMFAs which are too low. If the % prevalence of infection in mosquitoes is lower, then more mosquitoes should be dissected for examining the TBA of the antibodies. Figure 5D. DAPI signal is observed outside the parasite stages shown. Please provide better or cropped images. Line 81-89: Appropriate references should be cited for work done on TBV candidates i.e. Pf48/45, Pfs25, Pfs28 and PfHAP2.
Reviewer #2 (Remarks to the Author): Huang et al. report on a new potential vaccine candidate against malaria that elicits immunity against both the parasite's pre-erythrocytic and transmission stages. The vaccine combines the CSP preerythrocytic antigen and a fragment of the Pfs230 sexual stage antigen (Pf230D1) in immunogenic liposomes and 3-component adjuvant system. The authors show that immunization with these liposomes elicits the production of antibodies against both CSP and Pf230D1 that bind sporozoites and gametocytes, respectively, and functionally inhibit hepatic and mosquito infection, also respectively. The manuscript is clear and the conclusions are supported by the data. The results are of added value for the community and deserve publication. However, some aspects do need to be addressed beforehand, as detailed below.
-The Introduction covers the most important aspects of the topic at hand. However, I think the order in which the information is provided is not necessarily the most appropriate. Specifically, the section pertaining pre-erythrocytic vaccines goes somewhat back and forth, starting with a mention of CSP, followed by RTS,S and then R21, then explaining what the latter it is composed of, then back to explaining the domains in CSP and the immune responses it elicits, and ending with the composition of RTS,S. This seems rather illogical and a bit all over the place. Why not start by explaining what CSP is and does, detailing what immune responses it can elicit, then introduce RTS,S explaining how it is designed, followed by R21 and its design, possibly ending with the remaining CSP-based subunit vaccines mentioned in this paragraph? -Sentence "Pfs230 is a large protein containing over 3,000 amino acids (aa), fragments of which have been assessed as candidate TBV antigens." should be appropriately referenced.
-The authors should explain more clearly the rationale for the selection of residues 552-731 of Pfs230 for the construction of Pfs230D1. Are these residues chosen taking in account genetic variation i.e. was a more conserved region chosen to incorporate on the surface of liposomes? Why do these differ from the 542-736 aa stretch in Pfs230D1M?
-The data in Fig. 4 are very nice and informative as to the humoral immunogenicity of the bivalent antigens with the CoPoP liposomes. However, it would be nice to have a functional assay for the anti-CSP antibodies, similarly to what the authors did for the Pfs230 antibodies using SMFA. This is important to show that the anti-CSP antibodies elicited by immunization are indeed functional in inhibiting hepatic infection by P. falciparum. Such evaluation could be performed in vitro through Invasion Inhibition Assays employing HC04 cells or human primary hepatocytes, or in vivo, by passively transferring IgGs from the immunized animals into liver-humanized mice prior to challenge of these mice with P. falciparum sporozoites delivered by mosquito bite. The same applies to the data in Fig. 5.
-The order of the subsequent Results sections makes little sense to me. Why outbred mice for measurement of immune responses, then rabbits for passive transfer and challenge experiments, then inbred mice for challenge studies, then rabbits again for measurement of immune responses? Why don't the authors follow a logical order and describe their results on the immunogenicity in outbred mice (first the measurement of humoral and cellular responses, then the functionality by SMFA and challenge studies), then inbred mice (first the measurement of humoral and cellular responses, then the functionality by SMFA and challenge studies), and then do the same for the rabbits (first the measurement of humoral and cellular responses, then the functionality by SMFA and passive transfer + challenge studies)? As it is, in section "Antibody responses induced by bivalent, liposome-displayed CSP and Pfs230D1+ in inbred mice", the authors claim that they assessed vaccination in C57BL/6 mice prior to PbPfCSP sporozoite challenge but they do not present any data on the hepatic infection of immunized vs non-immunized mice after challenge with PbPfCSP sporozoites. In fact, it is not until section "Bivalent, liposome-displayed CSP and Pfs230D1+ induce protective immune responses" that PbPfCSP sporozoites were employed to challenge mice into which rabbit immune serum was passively transferred, and then to challenge inbred immunized mice with PbPfCSP sporozoites. The whole order in which these data are presented makes no sense to me.
-It is unclear to me why the authors initially resorted to the rabbit model and passive transfer of rabbit serum instead of simply challenging the immunized inbred mice, as they later did. -The authors do not discuss the innovation of the development of a Pfs230D1+/PfCSP vaccine in a field context. The discussion should also address the genetic diversity of the antigens in question.
- Supplementary Fig S2 and S3 -% inhibition of TRA: The authors should consider increasing the number of mosquitoes in these experiments. As Medley et al suggested, using less than 50-100 mosquitoes might provide unreliable estimates of TRA. Alternatively, the exact same experimental conditions should be replicated at least 3 times.
- Supplementary Fig S3: days 8 later should be 8 days later. Figure 7C, 8C: yy axis should mention which genes were involved in the assessment of the inhibition of parasite burden, otherwise, without reading the legend, it is not clear whether this was done by qPCR or IFA.

Response to Reviewer #1
The article by Huang et al. provides detailed analysis of CoPoP liposomes admixed with two malaria antigens Pfs230 and PfCSP for their transmission/infection blocking activity. These liposomes are stable at 37C, confirmational integrity of antigens is intact and can elicit antigen specific cellular immune response in the animals. These finding represent significant advance by targeting more than one Plasmodium life cycle stages.
Author Response: Thank you for the encouraging assessment.
Change "supporting" table# to "Supplementary "table throughout the manuscript.   Table 3. Only 20 mosquitoes were dissected per experiment in SMFAs which are too low. If the % prevalence of infection in mosquitoes is lower, then more mosquitoes should be dissected for examining the TBA of the antibodies.
Author Response: We and other have shown that TBA values fluctuate depending on mean oocyst in the controls even when the same sample is tested. In other words, it is not feasible to compare TBA data accurately if samples are tested in different assays/studies with different mean control oocysts. More importantly, we have shown that TBA is determined by TRA and mean control oocysts both in SMFA 1 and DMFA 2 . Therefore, we limit our focus to TRA in this work, by which people can meaningfully compare SMFA activity, which would not be the case for TBA.
For the number of dissected mosquitoes (n=20), we report 95%CI of TRA estimates (using mathematical modeling as described 3 ). In this way, readers can see uncertainly of each of TRA estimate under the current test condition. In general, it is true that n=40 assay has more power to detect a small difference than n=20 assay. But the important point is that even in this relatively "underpowered" assay (n=20), we still see statistically significant inhibitions, meaning the functional activities of anti-Pfs230 antibodies were strong. For the reviewer's reference, we have evaluated the power systematically in a previous study 4 . As shown in Fig 6 of that work, there is only minor difference in power between n=60 assay and n=20 assay as far as TRA>50%.
In addition, we note to the referee that the use of n=20 mosquitos in SMFA analytical testing is precedented in recent early-phase human clinical TBV trials. [5][6][7][8] References:   Huang et al. report on a new potential vaccine candidate against malaria that elicits immunity against both the parasite's pre-erythrocytic and transmission stages. The vaccine combines the CSP pre-erythrocytic antigen and a fragment of the Pfs230 sexual stage antigen (Pf230D1) in immunogenic liposomes and 3-component adjuvant system. The authors show that immunization with these liposomes elicits the production of antibodies against both CSP and Pf230D1 that bind sporozoites and gametocytes, respectively, and functionally inhibit hepatic and mosquito infection, also respectively. The manuscript is clear and the conclusions are supported by the data. The results are of added value for the community and deserve publication. However, some aspects do need to be addressed beforehand, as detailed below.
Author Response: Thank you for this assessment.
The Introduction covers the most important aspects of the topic at hand. However, I think the order in which the information is provided is not necessarily the most appropriate. Specifically, the section pertaining pre-erythrocytic vaccines goes somewhat back and forth, starting with a mention of CSP, followed by RTS,S and then R21, then explaining what the latter it is composed of, then back to explaining the domains in CSP and the immune responses it elicits, and ending with the composition of RTS,S. This seems rather illogical and a bit all over the place. Why not start by explaining what CSP is and does, detailing what immune responses it can elicit, then introduce RTS,S explaining how it is designed, followed by R21 and its design, possibly ending with the remaining CSP-based subunit vaccines mentioned in this paragraph?
Author Response: Thank you for the guidance. As suggested, we have re-ordered the introduction as suggested above.
Sentence "Pfs230 is a large protein containing over 3,000 amino acids (aa), fragments of which have been assessed as candidate TBV antigens." should be appropriately referenced. Fig  S3:  days  8  later  should  be  8  days later. Figure 7C, 8C: yy axis should mention which genes were involved in the assessment of the inhibition of parasite burden, otherwise, without reading the legend, it is not clear whether this was done by qPCR or IFA.

Supplementary
Author Response: Thank you again for your careful reading. We corrected the Fig S3  caption. For Figure 7C and 8C, we have revised the axis legend to indicate the measurement is liver qPCR based and we revised the figure caption to indicate inhibition was determined by using qPCR to assess parasite 18S rRNA relative to mouse GAPDH mRNA, compared to non-immunized control mice.